Affinity purification of ubiquitin-protein ligase on immobilized protein substrates. Evidence for the existence of separate NH2-terminal binding sites on a single enzyme.
Previous studies have indicated the existence of separate binding sites of ubiquitin-protein ligase, E3, specific for basic (Type I) or bulky hydrophobic (Type II) NH2-terminal amino acid residues of proteins. Another class (Type III) of protein substrates appeared to interact with E3 at regions other than the NH2 terminus (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698). In the present study we have used affinity chromatography on immobilized protein substrates to examine the question of whether the different binding sites belong to one E3 enzyme, or to different E3 species. Another objective was to develop a procedure for the extensive purification of E3. When a crude extract of reticulocytes is applied to Type I or Type II protein substrates linked to Sepharose, E3 becomes strongly bound to the affinity columns and is not eluted with salt at high concentration. However, the enzyme can be specifically eluted by a dipeptide that has an NH2-terminal residue similar to that of matrix-bound protein substrate. A 350-fold purification is obtained in this single step. Preparations of E3 purified on either Type I or Type II protein substrate affinity columns act on both types of protein substrates, indicating that the separate binding sites for basic and hydrophobic NH2-terminal residues belong to one enzyme. Another species of E3 that acts strongly on some Type III protein substrates does not bind to Type I or Type II protein substrate affinity columns.
Reiss
Y
Y
Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Hershko
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D004151:Dipeptides; D008025:Ligases / Q000097:blood / Q000302:isolation & purification*; D008962:Models, Theoretical; D011506:Proteins; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases
D004151:Dipeptides; D011506:Proteins; D044767:Ubiquitin-Protein Ligases; D008025:Ligases